Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways.
Ontology highlight
ABSTRACT:
SUBMITTER: Delaney JR
PROVIDER: S-EPMC1500970 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has largely been studied in cultured cells, and its in vivo function is not fully understood. We have isolated null mutations in the Drosophila dTAK1 gene that encodes dTAK1, a homolog of TAK1. dTAK1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Our genetic studies also indicate that dTAK1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, our epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. Our genetic demonstration of dTAK1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions.
| S-EPMC524699 | biostudies-literature
Project description:Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappaB-bound IkappaBalpha. Free IkappaBalpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappaB more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappaB activation; they have significant amounts of nuclear NF-kappaB. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappaB upon stimulation. These data demonstrate clearly that the coupled folding and binding of IkappaBalpha is critical for its precise control of NF-kappaB transcriptional activity.
| S-EPMC2519148 | biostudies-literature
Project description:Alpha-momorcharin (α-MMC), a member of the ribosome-inactivating protein (RIP) family, has been found in the seeds of Momordica charantia (bitter melon). α-MMC contributes a number of pharmacological activities; however, its inflammatory properties have not been well studied. Here, we aim to determine the inflammatory responses induced by recombinant α-MMC and identify the underlying mechanisms using cell culture and animal models. Recombinant α-MMC was generated in Rosetta™(DE3)pLysS and purified by the way of nitrilotriacetic acid (NTA) chromatography. Treatment of recombinant α-MMC at 40 μg/mL exerted sub-lethal cytotoxic effect on THP-1 monocytic cells. Transcriptional profiling revealed that various genes coding for cytokines and other proinflammatory proteins were upregulated upon recombinant α-MMC treatment in THP-1 cells, including MCP-1, IL-8, IL-1β, and TNF-α. Recombinant α-MMC was shown to activate IKK/NF-κB and JNK pathways and the α-MMC-induced inflammatory gene expression could be blocked by IKKβ and JNK inhibitors. Furthermore, murine inflammatory models further demonstrated that α-MMC induced inflammatory responses in vivo. We conclude that α-MMC stimulates inflammatory responses in human monocytes by activating of IKK/NF-κB and JNK pathways, raising the possibility that consumption of α-MMC-containing food may lead to inflammatory-related diseases.
| S-EPMC6949964 | biostudies-literature
Project description:BackgroundStephania yunnanensis H. S. Lo is widely used as an antipyretic, analgesic and anti-inflammatory herbal medicine in SouthWest China. In this study, we investigated the anti-inflammatory activity and mechanism of sinoacutine (sino), one of the primary components extracted from this plant.MethodsA RAW264.7 cell model was established using lipopolysaccharide (LPS) induced for estimation of cytokines in vitro, qPCR was used to estimate gene expression, western blot analysis was used to estimate protein level and investigate the regulation of NF- κB, JNK and MAPK signal pathway. In addition, an acute lung injury model was established to determine lung index and levels of influencing factors.ResultsUsing the RAW264.7 model, we found that sino reduced levels of nitric oxide (NO), tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and prostaglandin E2 (PGE2) but increased levels of IL-6. qPCR analysis revealed that sino (50, 25 μg/ml) inhibited gene expression of nitric oxide synthase (iNOS). western blot analysis showed that sino significantly inhibited protein levels of both iNOS and COX-2. Further signalling pathway analysis validated that sino also inhibited phosphorylation of p65 in the NF-κB and c-Jun NH2 terminal kinase (JNK) signalling pathways but promoted the phosphorylation of extracellular signal regulated kinase (ERK) and p38 in the MAPK signalling pathway. In addition, in a mouse model induced by LPS, we determined that sino reduced the lung index and the levels of myeloperoxidase (MPO), NO, IL-6 and TNF-α in lung tissues and bronchoalveolar lavage fluid (BALF) in acute lung injury (ALI).ConclusionTaken together, our results demonstrate that sino is a promising drug to alleviate LPS-induced inflammatory reactions.
| S-EPMC8605577 | biostudies-literature
Project description:The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. Here we provide genetic evidence that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. We also demonstrate that Dif is a downstream component of the Toll signaling pathway in activating the drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development.
| S-EPMC316597 | biostudies-literature
Project description:The intensity and duration of macrophage-mediated inflammatory responses are controlled by proteins that modulate inflammatory signaling pathways. MCPIP1 (monocyte chemotactic protein-induced protein 1), a recently identified CCCH Zn finger-containing protein, plays an essential role in controlling macrophage-mediated inflammatory responses. However, its mechanism of action is poorly understood. In this study, we show that MCPIP1 negatively regulates c-Jun N-terminal kinase (JNK) and NF-?B activity by removing ubiquitin moieties from proteins, including TRAF2, TRAF3, and TRAF6. MCPIP1-deficient mice spontaneously developed fatal inflammatory syndrome. Macrophages and splenocytes from MCPIP1(-/-) mice showed elevated expression of inflammatory gene expression, increased JNK and I?B kinase activation, and increased polyubiquitination of TNF receptor-associated factors. In vitro assays directly demonstrated the deubiquitinating activity of purified MCPIP1. Sequence analysis together with serial mutagenesis defined a deubiquitinating enzyme domain and a ubiquitin association domain in MCPIP1. Our results indicate that MCPIP1 is a critical modulator of inflammatory signaling.
| S-EPMC3005225 | biostudies-literature
Project description:Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-kappaB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3' UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.
| S-EPMC1567904 | biostudies-literature
Project description:NF-kappaB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-kappaB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-kappaB and IkappaB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.
| S-EPMC2701504 | biostudies-literature
Project description:NF-kappaB is a pleiotropic transcription factor with key functions in the intestinal immune system. NF-kappaB family members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation. The perpetuated activation of NF-kappaB in patients with active inflammatory bowel disease suggests that regulation of NF-kappaB activity is a very attractive target for therapeutic intervention. Such strategies include antioxidants, proteasome inhibitors, inhibition of NF-kappaB by adenoviral I kappaB alpha expression vectors, and antisense DNA targeting of NF-kappaB. These approaches will hopefully permit the design of new treatment strategies for chronic intestinal inflammation.
| S-EPMC1727350 | biostudies-other
Project description:AimTo study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed.MethodspECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.ResultsCells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis.ConclusionsIkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.
| S-EPMC4125672 | biostudies-literature